Screening using S. cerevisiae mannosyltransferase encoding genes

Abstract

The present invention relates to an antifungal in vitro and in vivo screening assays for identifying compounds which inhibit mannosyltransferases involved in protein O- and N-glycosylation. The antifungal screening assay for identifying a compound which inhibit mannosyltransferases involved in protein O- and N-glycosylation, comprises the steps of: a) subjecting proteins to a specific mannosyltransferase protein encoded by a Saccharomyces cerevisiae mannosyltransferase encoding gene, wherein said gene is selected from the group consisting of KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7; b) subjecting step a) to a screened compound and determining the absence or presence of protein O- and N-glycosylation, wherein the absence of protein O- and N-glycosylation is indicative of an antifungal compound. There is also disclosed an in vitro method for the diagnosis of diseases caused by fungal infection in a patient.

What is claimed is: 
     
       1. An antifungal screening assay for identifying compounds that inhibit mannosyltransferases involved in protein O- and N-linked glycosylation, which comprises the steps of: a) providing a mannosyltransferase protein encoded by a Saccharomyces cerevisiae mannosyltransferase encoding gene selected from the group consisting of KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7 and further providing a specific substrate of said mannosvltransferase protein;   b) subjecting the mannosyltransferase protein and specific substrate of step a) to a compound suspected of having antifungal activity and determining absence or presence on said substrate of specific O- and N-linked oligosaccharides, wherein absence of at least one of said oligosaccharides is indicative of an antifungal compound that specifically inhibits at least one member of the group of step a).   
     
     
       2. An in vivo antifungal screening assay for identifying compounds that inhibit mannosyltranferases involved in protein O- and N-linked glycosylation, which comprises the steps of: a) separately cultivating a mutant yeast strain containing at least one gene encoding a mannosyltransferase wherein said gene has been inactivated and a control yeast strain containing said at least one gene;   b) subjecting said both yeast strains of step a) to a compound suspected of having antifungal activity; and   c) determining if said compound selectively inhibits growth of said control yeast strain but not of said mutant yeast strain, wherein the selective inhibition of growth is indicative of an antifungal compound that inhibits a mannosyltransferase involved in protein O- and N-linked glycosylation.   
     
     
       3. The method of claim 2, wherein said gene is selected from the group consisting of KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7. 
     
     
       4. An in vivo antifungal screening assay for identifying compounds that elicit a reporter gene response in yeast equivalent to that generated by yeast strains carrying a defect in a Ktrp mannosyltranferase, which comprises the steps of: a) cultivating a yeast strain having a diagnostic set of reporter genes for a Ktrp mannosyltransferase defect caused by a mutation in the gene encoding said mannosyltransferase;   b) subjecting said yeast strain of step a) to a compound suspected of having antifungal activity and determining if said compound selectively elicits a response from said reporter genes equivalent to that generated by yeast strains carrying a defect in a Ktrp mannosyltranferase, wherein said response is indicative of a compound being a candidate for a specific antifungal inhibitor.   
     
     
       5. An in vitro method for the diagnosis of diseases caused by fungal infection in a patient, which com- prises the steps of: a) obtaining a biological sample from said patient;   b) subjecting said sample to PCR using a primer pair specific for a mannosyltransferase gene of Candida albicans homolog to a gene selected from the group consisting of KRE2/1MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7, wherein a presence of said gene is indicative of the presence of fungal infection.   
     
     
       6. The method of claim 5, wherein said fungal infection is caused by Candida. 
     
     
       7. An in vitro method for the diagnosis of diseases caused by fungal infection in a patient, which comprises the steps of: a) obtaining a biological sample from said patient;   b) subjecting said sample to an antibody specific for a specific Ktrp mannosyltransferase antigen, wherein a presence of said antigen is indicative of the presence of fungal infection.   
     
     
       8. The method of claim 7, wherein said fungal infection is caused by Candida.